The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.
Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.
Synaptic dysfunction is among the earliest known hallmarks of most major neurodegenerative diseases1,2,3,4,5,6. However, very little is known regarding how these structural and functional impairments relate to later stages of disease progression. Drosophila has proven to be a useful model system for understanding synapse growth and development using larval NMJs7,8,9. However, the third larval instar stage only lasts a few days, limiting their utility in studying progressive, age-dependent neurodegeneration. An alternative to assessing larval NMJs is to examine synaptic structures in adult Drosophila, such as the synapses formed on the Dorsal Longitudinal Muscles (DLMs) that are required for flight10,11,12,13,14,15,16. These tripartite synapses are structurally organized in a similar manner to mammalian synapses17, providing a unique advantage for assessing models of neurodegenerative diseases.
Here we describe a straightforward method for analyzing the structural integrity of adult NMJs in a Drosophila model of neurodegeneration. Previous DLM dissection methods and studies have emphasized the importance of preserving muscle tissue for a variety of applications18,19,20,21,22,23. Our protocol provides a comprehensive method to preserve both neuronal and muscle tissue to investigate neurodegenerative diseases. Another major component of studying these diseases is the ability to understand neuronal loss in an age dependent manner. Previous work provides a critical and in-depth understanding of how the DLM NMJs are formed during metamorphosis into early adulthood11,12,14,15,16,24. Our protocol establishes a method to build upon this work to investigate DLM NMJs in an age-dependent manner in aging and neurodegenerative diseases.
1. Generation of transgenic flies
2. Dissection prep
3. Thorax isolation and fixation
4. Flash freezing and thorax bisection
5. Structural staining
6. Mounting tissue
7. Alternative: Staining with primary antibodies
NOTE: This section is optional and should be used directly between sections 4 and 5 if desired.
The generation of transgenic flies expressing human Tar-Binding Protein of 43 kDa mutant (TDP-43M337V) is represented by the schematic (Figure 1A). This demonstrates the application of the binary Gal4/UAS system in Drosophila27. The illustration depicts a hemithorax with six muscle fibers, A‒F going from the most dorsal fiber A to the most ventral F (Figure 1B)11,12. To assess synaptic integrity, NMJs were stained with HRP and Phalloidin (Figure 1C‒E). Motor neurons in TDP-43M337V mutants (Figure 1F) have little to no HRP staining by Day 21, while WT (Oregon-R) remains intact (Figure 1C). There are no visible differences in muscle staining (Figure 1D,G). The changes in gross morphology observed in TDP-43M337V mutants demonstrates how synaptic integrity can be implicated in a neurodegenerative disease model of amyotrophic lateral sclerosis (ALS) using the adult DLM model. In addition to structural staining, staining the DLM NMJs can also provide an assessment of synaptic integrity with presynaptic (Figure 2A‒R) and post synaptic (Figure 2S‒X) markers. Together, these results illustrate how this dissection protocol could be applied to studying DLM tissue in neurodegenerative diseases.
One key aspect of this dissection is the application of liquid nitrogen to flash freeze the tissue to make the bisection easier. The utility of the liquid nitrogen is demonstrated in WT flies with liquid nitrogen where muscle tissue has no damage or nicked fibers (Figure 3A‒C). Without liquid nitrogen, the tissue can be more difficult to dissect. For example, following this protocol and skipping the liquid nitrogen flash freezing step allows the tissue to be more susceptible to damage from the dissection tools such as damaged neurons (Figure 3D) or damaged muscle fibers (Figure 3E). The application of liquid nitrogen helps to prevent tissue damage that could occur when working with DLM tissue regardless of the genotype of the specimen (Figure 3C and 3F).
Figure 1: Progressive denervation of DLM synapses in a Drosophila model of ALS. (A) The generation of ALS transgenic flies expressing a human mutant form of Tar-Binding Protein of 43 kDa (TDP-43) are shown in the schematic. (B) The illustration depicts the shape and orientation of a hemithorax in an adult Drosophila. Using the protocol, we can observe the progressive loss of synaptic integrity of DLM NMJ synapses through structural staining of motor neurons with HRP (green) and muscle tissue with Phalloidin (magenta). Our model depicts the loss of synaptic integrity in an adult model of ALS through the generation of adult flies expressing a mutant from of human TDP-43M337V in motor neurons (Figure 1F‒H) in comparison to WT (Figure 1C‒E) flies in muscle fiber C. Arrows highlight examples of a WT synapse (Figure 1C) and an example of loss of synaptic integrity. Scale bar =20 µm at 63x magnification. Please click here to view a larger version of this figure.
Figure 2: Assessing synaptic integrity using presynaptic markers at adult NMJs. Synaptic integrity can also be assessed using presynaptic and postsynaptic markers in WT flies that are 14 days old in muscle fiber C. The presynaptic markers Synapsin (B), Syntaxin (H), and Bruchpilot (BRP) (N) are co-stained with HRP (A, G, M). The staining depicts the localization of these markers to the presynaptic terminals (C, I, O). At higher magnification, the images illustrate the localization of Synapsin (E), Syntaxin (K), and BRP (Q) with HRP (D, J, and P) in more detail (Figure F, L, and R). We also show a postsynaptic marker Glutamate Receptor III (GluRIII) (T) co-stained with HRP (S). The co-staining demonstrates the utility of these markers (U). At higher magnification the representative images exemplify the localization (X) of GluRIII (W) and HRP (V) to the postsynaptic muscle tissue and the presynaptic terminals, respectively. Scale bar for panels A‒C, G-I, M‒O, S‒U represent 20 µm at 63x magnification. Scale bar for panels D‒F, 2J-2L, 2P-2R, and 2V-2X represent 10 µm at 63x magnification. Please click here to view a larger version of this figure.
Figure 3: Utility of liquid nitrogen for DLM dissections. To demonstrate the utility of liquid nitrogen for the DLM dissections, we show a comparison of day 21 WT flies with and without liquid nitrogen from muscle fiber C. With liquid nitrogen, Phalloidin (B) remains intact and does not compromise the HRP staining (A, C). Without liquid nitrogen, muscle tissue becomes stringy and difficult to bisect (E) and HRP staining (D, F) becomes compromised due to technical error. White arrows show an area of no muscle damage in with liquid nitrogen (B) and damaged muscle tissue (E). Scale bar = 20 µm at 63x magnification. Please click here to view a larger version of this figure.
Using the methods described in this protocol, we provide a straightforward approach for dissection of the DLM tissue and demonstrate how this can be applied to assess synaptic integrity through structural staining and synaptic markers in adult Drosophila. One critical step in the protocol that makes the DLM tissue easier to dissect is the flash freezing with liquid nitrogen. Without this step, the tissue is less firm and more difficult to cut precisely as observed in Figure 3. This protocol builds upon previous dissection methods to allow the preservation of both motor neurons and muscle tissue18,19,20,21,22,23. One limitation of this protocol is that when making the cut down the midline for the bisection, it can be difficult to get two clean preps per thorax. One way to ensure at least one hemithorax per fly, you can purposely cut off to one side of the thorax to get one clean prep. With this modification, one may also need to remove additional excess tissue from the cut to clean up the sample with the blade breaker. For those new to this technique, with continued practice, accuracy of the bisection will increase.
The method described here allows researchers to easily assess structural integrity of adult DLM NMJs at any time throughout their lifespan. A major advantage of this protocol is the ability to access synaptic integrity in neurodegenerative disease models by using synaptic markers. We demonstrate that this application can help visualize changes in gross morphology with structural staining (Figure 1C‒H). Additionally, synaptic integrity can be assessed with staining of presynaptic markers including but not limited to Synapsin28 (Figure 2A‒F), Syntaxin29 (Figure 2G‒L) and BRP30 (Figure 2M‒R). The postsynaptic muscle tissue can also be assessed using the Glutamate Receptor III subunit antibody31 (Figure 2S‒X), demonstrating the utility of this protocol.
Researchers can also utilize this dissection method to complement functional data to comprehensively examine the structural integrity of synapses associated with a wide variety of diseases. These synapses also allow for functional analysis through electrophysiological recordings32,33,34 and the flight assay10. This protocol can also provide ease of access to the tissue for many applications and assays. Future studies, for example, could use this protocol to quantify synaptic changes through quantification of the density and number of synapses15,16. While the protocol described here specifically examines synaptic integrity of motor neurons, complementary protocols for assessing muscle cell loss can also be performed with this dissection using TUNEL staining35. To examine neuronal loss, dissection of the thoracic ganglion36 could also be used with TUNEL staining. We expect that the dissection described here will have more applications to future studies assessing age-related pathologies as well as neurodegenerative diseases.
The authors have nothing to disclose.
This work was supported by the National Institutes of Health (R01 NS110727) to D.T.B.
32% Formaldehyde | Electron Microscopy Sciences | 15714 | Tissue preservation |
Alexa Fluor 568 goat anti mouse | Fisher Scientific | A11031 | Labels primary antibodies. Used at 1:200 concentration. |
Alexa Fluor 568 goat anti rabbit | Fisher Scientific | A11036 | Labels primary antibodies. Used at 1:200 concentration. |
anti- Bruchpilot (BRP) antibody | Developmental Studies Hybridoma Bank | NC82 | Stains the active zones in presynaptic neurons. Used at 1:25 concentration. |
anti-GluRIII antibody | Gift from Aaron DiAntonio | N/A | Labels glutamate receptor subunits. Used at 1:1000 concentration. |
anti-Synapsin antibody | Developmental Studies Hybridoma Bank | 3C11 | Labels the synaptic protein synapsin. Used at 1:50 concentration. |
anti-Syntaxin antibody | Developmental Studies Hybridoma Bank | 8C3 | labels the synaptic protein syntaxin. Used at 1:10 concentration. |
BenchRocker | Genesee Scientific | 31-302 | Rotating samples during staining |
Blade Breaker | Fine Science Tools | 10053-09 | Used for holding feather blade |
cover slips | Fisher Scientific | 12548A | For mounting tissue |
cryogenic gloves | VWR | 97008-198 | protect hands from liquid nitrogen |
cryogenic tweezers | VWR | 82027-432 | Hold 2.0 mL tube in liquid nitrogen |
dewar flask-1900 mL | Thomas Scientific | 5028M54 | Hold liquid nitrogen |
Feather Blades | Electron Microscopy Sciences | 72002-01 | Scalpel Blades |
Fine Forecps x 2 | Fine Science Tools | 11252-20 | One fine pair for Clearing midline of thorax. The other pair can be dulled using a sharpening stone. |
FITC-conjugated anti HRP | Jackson Laboratories | 123-545-021 | Stains Motor Neurons. Used at 1:100 concentration |
freezer box (Black) | Fisher Scientific | 14100F | Protects samples from light |
glass pasteur pipettes | VWR | 14637-010 | Used to transfer samples |
glass slides | Fisher Scientific | 12550143 | For mounting tissue |
mounting media (vectashield) anti-fade | VWR | 101098-042 | Mounting media retains fluorescent signaling |
nail polish | Electron Microscopy Sciences | 72180 | Seals microscope slides |
normal goat serum | Fisher Scientific | PCN5000 | Prevents non-specific binding of antibodies |
paint brush | Genesee Scientific | 59-204 | Transferring flies |
PBS | Fisher Scientific | 10-010-023 | Saline solution for dissecting and staining |
Phalloidin 647 | Abcam | AB176759 | Stains F-Actin in muscle Tissue. Used at 1:1000 concentration |
plastic petri dish (100 mm) | VWR | 25373-100 | Dissection dish |
reinforcement labels | W.B. Mason | AVE05722 | Provides support for glass coverslip over the mounted tissue |
sharpening block | Grainger | 1RDF5 | Keeping fine forceps sharp and also dulling separate pair |
slide folder | VWR | 10126-326 | Sample storage |
standard office scissors | W.B. Mason | ACM40618 | Cutting reinforcement labels |
Sylgard 184 | Electron Microscopy Sciences | 24236-10 | Coating for dissection dish |
Triton-X-100 | Electron Microscopy Sciences | 22140 | Helps to permeabilize tissue |
Vannas Disssection Sissors | Fine Science Tools | 1500-00 | Ued for removing fly legs and making an incision on thorax |